Figure 3.

Lower levels of Th17 cells and higher levels of Tregs were observed in the spleen of Cd226^−/− EAE mice. (A) The plasma levels of IFN-γ and IL-10 in Cd226^−/− and WT mice during EAE pathogenesis were assessed by ELISA (n = 8). (B) Protein expression of IFN-γ, IL-4, IL-17A and IL-10 in sorted CD4⁺ T cells from splenocytes in Cd226^−/− mice and WT littermates was assessed by intracellular staining. The numbers in each quadrant show the percentage of the relevant cell population (n = 8). (C) Splenocytes were stimulated in vitro with PMA (phorbol myristate acetate), ionomycin and brefeldin A in 10% FBS/RPMI 1,640 for 6 h. FCM data analysis of CD4⁺ T cell subsets for cytokines released from the spleens of Cd226^−/− or WT mice was conducted on days 15–18 after immunization with MOG₃₅₋₅₅ (gated on total CD4⁺ T cells, with frequencies on the left). The proportions of Th1, Th2, Th17 cells and Tregs were determined by intracellular cytokine staining (n = 6). (D) The relative mRNA levels of IFN-γ, IL-4, IL-17A, and IL-10 in CD4⁺ T cells isolated from the spleens of Cd226^−/− or WT mice at days 15–18 after immunization with MOG₃₅₋₅₅ were determined by RT-qPCR (n = 6). (E) The relative mRNA levels of Foxp3 and RORγt in CD4⁺ T cells isolated from the splenocytes of WT or Cd226^−/− mice on days 15–18 after immunization with MOG₃₅₋₅₅ were determined by RT-qPCR (n = 6). The results represent at least three independent experiments (A–E). The data are presented as the mean ± SEM. ^nsP > 0.05, *P < 0.05, **P < 0.005, ***P < 0.001.